Mutant α-amylase

ABSTRACT

Novel α-amylase enzymes are disclosed in which one or more asparagine residues are substituted with a different amino acid or deleted. The disclosed α-amylase enzymes show altered or improved low pH starch hydrolysis performance, stability and activity profiles.

FIELD OF THE INVENTION

The present invention is directed to α-amylases having altered performance characteristics. The present invention is also directed to novel mutant α-amylase enzymes having at least an asparagine residue which is substituted with a different amino acid or deleted, wherein the resultant α-amylase exhibits altered low pH starch hydrolysis performance, altered stability and altered activity profiles.

BACKGROUND OF THE INVENTION

α-Amylases (α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) hydrolyze internal α-1,4-glucosidic linkages in starch, largely at random, to produce smaller molecular weight malto-dextrins. α-Amylases are of considerable commercial value, being used in the initial stages (liquefaction) of starch processing; in alcohol production; as cleaning agents in detergent matrices; and in the textile industry for starch desizing. α-Amylases are produced by a wide variety of microorganisms including Bacillus and Aspergillus, with most commercial amylases being produced from bacterial sources such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, or Bacillus stearothermophilus. In recent years, the preferred enzymes in commercial use have been those from Bacillus licheniformis because of their heat stability and performance, at least at neutral and mildly alkaline pH's.

In general, starch to fructose processing consists of four steps: liquefaction of granular starch, saccharification of the liquefied starch into dextrose, purification, and isomerization to fructose. The object of a starch liquefaction process is to convert a concentrated suspension of starch polymer granules into a solution of soluble shorter chain length dextrins of low viscosity. This step is essential for convenient handling with standard equipment and for efficient conversion to glucose or other sugars. To liquefy granular starch, it is necessary to gelatinize the granules by raising the temperature of the granular starch to over about 72° C. The heating process instantaneously disrupts the insoluble starch granules to produce a water soluble starch solution. The solubilized starch solution is then liquefied by α-amylase (EC 3.2.1.1.).

A common enzymatic liquefaction process involves adjusting the pH of a granular starch slurry to between 6.0 and 6.5, the pH optimum of α-amylase derived from Bacillus licheniformis, with the addition of calcium hydroxide, sodium hydroxide or sodium carbonate. The addition of calcium hydroxide has the advantage of also providing calcium ions which are known to stabilize the α-amylases against inactivation. Upon addition of α-amylases, the suspension is pumped through a steam jet to instantaneously raise the temperature to between 80-115° C. The starch is immediately gelatinized and, due to the presence of α-amylases, depolymerized through random hydrolysis of α(1-4) glycosidic bonds to a fluid mass which is easily pumped.

In a second variation to the liquefaction process, α-amylase is added to the starch suspension, the suspension is held at a temperature of 80-100° C. to partially hydrolyze the starch granules, and the partially hydrolyzed starch suspension is pumped through a jet at temperatures in excess of about 105° C. to thoroughly gelatinize any remaining granular structure. After cooling the gelatinized starch, a second addition of α-amylase can be made to further hydrolyze the starch.

A third variation of this process is called the dry milling process. In dry milling, whole grain is ground and combined with water. The germ is optionally removed by flotation separation or equivalent techniques. The resulting mixture, which contains starch, fiber, protein and other components of the grain, is liquefied using α-amylase. The general practice in the art is to undertake enzymatic liquefaction at a lower temperature when using the dry milling process. Generally, low temperature liquefaction is believed to be less efficient than high temperature liquefaction in converting starch to soluble dextrins.

Typically, after gelatinization the starch solution is held at an elevated temperature in the presence of α-amylase until a DE of 10-20 is achieved, usually a period of 1-3 hours. Dextrose equivalent (DE) is the industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. Unhydrolyzed granular starch has a DE of virtually zero, whereas the DE of D-glucose is defined as 100.

The maximum temperature at which the starch solution containing α-amylase can be held depends upon the microbial source from which the enzyme was obtained and the molecular structure of the α-amylase molecule. α-Amylases produced by wild type strains of Bacillus subtilis or Bacillus amyloliquefaciens are typically used at temperatures no greater than about 90° C. due to excessively rapid thermal inactivation above that temperature, whereas α-amylases produced by wild type strains of Bacillus licheniformis can be used at temperatures up to about 110° C. The presence of starch and calcium ion are known to stabilize α-amylases against inactivation. Nonetheless, α-amylases are used at pH values above 6 to protect against rapid inactivation. At low temperatures, α-amylase from Bacillus licheniformis is known to display hydrolyzing activity on starch substrate at pH values as low as 5. However, when the enzyme is used for starch hydrolysis at common jet temperatures, e.g., between 102° C. and 109° C., the pH must be maintained above at least pH 5.7 to avoid excessively rapid inactivation. The pH requirement unfortunately provides a narrow window of processing opportunity because pH values above 6.0 result in undesirable by-products, e.g., maltulose. Therefore, in reality, liquefaction pH is generally maintained between 5.9 and 6.0 to attain a satisfactory yield of hydrolyzed starch.

Another problem relating to pH of liquefaction is the need to raise the pH of the starch suspension from about 4, the pH of a corn starch suspension as it comes from the wet milling stage, to 5.9-6.0. This pH adjustment requires the costly addition of acid neutralizing chemicals and also requires additional ion-exchange refining of the final starch conversion product to remove the chemical. Moreover, the next process step after liquefaction, typically saccharification of the liquefied starch into glucose with glucoamylase, requires a pH of 4-4.5; therefore, the pH must be adjusted down from 5.9-6.0 to 4-4.5; requiring additional chemical addition and refining steps.

Subsequent to liquefaction, the processed starch is saccharified to glucose with glucoamylase. A problem with present processes occurs when residual starch is present in the saccharification mixture due to an incomplete liquefaction of the starch, e.g., inefficient amylose hydrolysis by amylase. Residual starch is highly resistant to glucoamylase hydrolysis. It represents a yield loss and interferes with downstream filtration of the syrups.

Additionally, many α-amylases are known to require the addition of calcium ion for stability. This further increases the cost of liquefaction.

In U.S. Pat. No. 5,322,778, liquefaction between pH 4.0 and 6.0 was achieved by adding an antioxidant such as bisulfite or a salt thereof, ascorbic acid or a salt thereof, erythorbic acid, or phenolic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, or α-tocopherol to the liquefaction slurry. According to this patent, sodium bisulfite must be added in a concentration of greater than 5 mM.

In U.S. Pat. No. 5,180,669, liquefaction between a pH of 5.0 to 6.0 was achieved by the addition of carbonate ion in excess of the amount needed to buffer the solution to the ground starch slurry. Due to an increased pH effect which occurs with addition of carbonate ion, the slurry is generally neutralized by adding a source of hydrogen ion, for example, an inorganic acid such as hydrochloric acid or sulfuric acid.

In PCT Publication No. WO 94/02597, a mutant α-amylase having improved oxidative stability is described wherein one or more methionines are replaced by any amino acid except cysteine or methionine.

In PCT publication No. WO 94/18314, a mutant α-amylase having improved oxidative stability is described wherein one or more of the methionine, tryptophan, cysteine, histidine or tyrosine residues is replaced with a non-oxidizable amino acid.

In PCT Publication No. WO 91/00353, the performance characteristics and problems associated with liquefaction with wild type Bacillus licheniformis α-amylase are approached by genetically engineering the α-amylase to include the specific substitutions Ala-111-Thr, His-133-Tyr and/or Thr-149-Ile.

Studies using recombinant DNA techniques to explore which residues are important for the catalytic activity of amylases and/or to explore the effect of modifying certain amino acids within the active site of various amylases and glycosylases have been conducted by various researchers (Vihinen et al., J. Biochem., vol. 107, pp. 267-272 (1990); Holm et al., Protein Engineering, vol. 3, pp. 181-191 (1990); Takase et al., Biochemica et Biophysica Acta, vol. 1120, pp. 281-288 (1992); Matsui et al., Febs Letters, vol. 310, pp. 216-218 (1992); Matsui et al., Biochemistry, vol. 33, pp. 451-458 (1992); Sogaard et al., J. Biol. Chem., vol. 268, pp. 22480-22484 (1993); Sogaard et al., Carbohydrate Polymers, vol. 21, pp. 137-146 (1993); Svensson, Plant Mol. Biol., vol. 25, pp. 141-157 (1994); Svensson et al., J. Biotech. vol. 29, pp. 1-37 (1993)). Researchers have also studied which residues are important for thermal stability (Suzuki et al., J. Biol. Chem. vol. 264, pp. 18933-18938 (1989); Watanabe et al., Eur. J. Biochem. vol. 226, pp. 277-283 (1994)); and one group has used such methods to introduce mutations at various histidine residues in a Bacillus licheniformis amylase, the rationale being that Bacillus licheniformis amylase which is known to be relatively thermostable when compared to other similar Bacillus amylases, has an excess of histidines and, therefore, it was suggested that replacing a histidine could affect the thermostability of the enzyme. This work resulted in the identification of stabilizing mutations at the histidine residue at the +133 position and the alanine residue at position +209 (Declerck et al., J. Biol. Chem., vol. 265, pp. 15481-15488 (1990); FR 2 665 178-A1; Joyet et al., Bio/Technology, vol. 10, pp. 1579-1583 (1992)).

Despite the advances made in the prior art, a need exists for an α-amylase which is effective enough at low pH values to allow commercial liquefaction at lower pH than currently practical. Similarly, a need exists in the art for a method which allows efficient liquefaction of dry milled grain at high temperatures. Further, a need exists in the art for a method which allows the efficient liquefaction of starch with a decreased reliance on the costly addition of calcium. Additionally, a need exists for a more efficient enzyme to effect a more complete hydrolysis of starch at the liquefaction stage to ensure efficient saccharification. Because commercially available amylases are not acceptable under many conditions due to stability problems, for example, the high alkalinity and oxidant (bleach) levels associated with detergents, there is a need for an amylase having altered, and preferably increased, performance profiles under such conditions. Thus, altered performance characteristics such as increased activity, thermostability, pH stability, oxidative stability or calcium stability which can be achieved while also altering, maintaining, or increasing enzymatic activity as compared to the wild type or precursor enzyme, would be desirable.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an α-amylase having altered performance profiles, such as pH stability, alkaline stability, oxidative stability or enzymatic activity.

It is a further object of the present invention to provide an α-amylase having increased stability in the absence of added calcium ion during liquefaction of starch.

It is a further object of the present invention to provide an α-amylase having altered low pH stability for use in efficient low pH liquefaction.

It is yet a further object of the present invention to provide an α-amylase which allows efficient liquefaction of dry milled grain at high temperatures.

It is still a further object of the present invention to provide an α-amylase which is useful in high pH environments or in the presence of oxidants or bleach.

It is still a further object of the present invention to provide an α-amylase which effects a more complete hydrolysis of starch molecules to increase the efficiency of saccharification.

According to the present invention, an α-amylase is provided that is the expression product of a mutated DNA sequence encoding an α-amylase, the mutated DNA sequence being derived from a precursor α-amylase by the deletion or substitution of one or more residues having the effect to improve the performance of the α-amylase residues.

Preferably, the deleted or substituted residue is an asparagine residue, most preferably at a position corresponding to N188 in Bacillus licheniformis. Where it is desired to alter the thermostability of the α-amylase, the asparagine substitution may be any other amino acid, including any of the 20 naturally occurring amino acids. Preferably, the substitution corresponds to N188S or N188T in Bacillus licheniformis. Also preferably, the α-amylase further comprises the deletion or substitution of a methionine or tryptophan residue, particularly at a position corresponding to M15, W138 and/or M197, or at a residue corresponding to V128, H133, S187 and/or A209 in Bacillus licheniformis. In a most preferred embodiment, an α-amylase is provided comprising substitutions at residues corresponding to M15L/N188S or M15T/N188S in Bacillus licheniformis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates mutagenic oligonucleotides useful during directed mutagenesis of Ans188 from Bacillus licheniformis α-amylase. In this and following figures illustrating oligonucleotide constructs, bold letters indicate base changes introduced by the oligonucleotide and underlining indicates restriction endonuclease sites introduced by the oligonucleotide.

FIG. 2 illustrates PCR primers used for PCR processing of mutagenic oligonucleotide templates.

FIG. 3 illustrates the DNA sequence of the gene for α-amylase from Bacillus licheniformis (NCIB 8061) (SEQ ID NO:33) and deduced amino acid sequence of the translation product (SEQ ID NO:41) as described by Gray et al., J. Bacteriology, vol. 166, pp. 635-643 (1986).

FIG. 4 illustrates the amino acid sequence (SEQ ID NO:34) of the mature α-amylase enzyme from Bacillus licheniformis.

FIG. 5 illustrates an alignment of the primary structures of three Bacillus α-amylases. The Bacillus licheniformis α-amylase (Am-Lich) (SEQ ID NO:35) is described by Gray et al., J. Bacteriology, vol. 166, pp. 635-643 (1986); the Bacillus amyloliquefaciens α-amylase (Am-Amylo) (SEQ ID NO:36) is described by Takkinen et al., J. Biol. Chem., vol. 258, pp. 1007-1013 (1983); and the Bacillus stearothermophilus α-amylase (Am-Stearo) (SEQ ID NO:37) is described by Ihara et al., J. Biochem., vol. 98, pp. 95-103 (1985).

FIG. 6 illustrates plasmid pHP13 wherein Cm^(R) refers to chloramphenicol resistance, Em^(R) refers to erythromycin resistance and Rep pTA1060 refers to the origin of replication from plasmid pTA1060.

FIG. 7 illustrates the pBLapr plasmid wherein BL AA refers to Bacillus licheniformis α-amylase gene; aprE refers to the promoter and signal peptide encoding region of the aprE gene; AmpR refers to the ampicillin resistant gene from pBR322; and CAT refers to the chloramphenicol resistance gene from pC194.

FIG. 8 illustrates the pHP.BL plasmid carrying the gene for Bacillus licheniformis α-amylase.

FIG. 9 illustrates a schematic of the PCR method used to produce the mutant oligonucleotides corresponding to α-amylase derived from Bacillus licheniformis.

FIG. 10 illustrates a graph derived from a statistical analysis of variant enzyme according to the invention, M15T/N188S, compared to wild type Bacillus licheniformis α-amylase in starch liquefaction at 107° C., 60 ppm calcium and varying pH.

FIG. 11 illustrates a graph derived from a statistical analysis of the performance of a variant enzyme according to the invention, M15T/N188S, compared to wild type Bacillus licheniformis α-amylase in starch liquefaction at 107° C., pH 6.0 and varying calcium concentration.

FIG. 12 illustrates a graph derived from a statistical analysis of the performance of a variant enzyme according to the invention, M15T/N188S, compared to wild type Bacillus licheniformis α-amylase in starch liquefaction at pH 6.0, 60 ppm calcium and varying temperature.

FIG. 13 illustrates the signal sequence-mature protein junctions in α-amylase derived from Bacillus licheniformis (SEQ ID NO:38), Bacillus subtilis aprE (SEQ ID NO:39) and Bacillus licheniformis in pBLapr (SEQ ID NO:40).

DETAILED DESCRIPTION

"α-Amylase" means an enzymatic activity which cleaves or hydrolyzes the α(1-4)glycosidic bond, e.g., that in starch, amylopectin or amylose polymers. α-Amylase as used herein includes naturally occurring α-amylases as well as recombinant α-amylases. Preferred α-amylases in the present invention are those derived from Bacillus licheniformis, Bacillus amyloliquefaciens or Bacillus stearothermophilus, as well as fungal α-amylases such as those derived from Aspergillus (i.e., A. oryzae and A. niger.

"Recombinant α-amylase" means an α-amylase in which the DNA sequence encoding the naturally occurring α-amylase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the α-amylase sequence compared to the naturally occurring α-amylase.

"Expression vector" means a DNA construct comprising a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome-binding sites, and sequences which control termination of transcription and translation. A preferred promoter is the Bacillus subtilis aprE promoter. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, plasmid and vector are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

"Host strain" or "host cell" means a suitable host for an expression vector comprising DNA encoding the α-amylase according to the present invention. Host cells useful in the present invention are generally procaryotic or eucaryotic hosts, including any transformable microorganism in which the expression of α-amylase according to the present invention can be achieved. Specifically, host strains of the same species or genus from which the α-amylase is derived are suitable, such as a Bacillus strain. Preferably, an α-amylase negative Bacillus strain (genes deleted) and/or an α-amylase and protease deleted Bacillus strain (ΔamyE, Δapr, Δnpr) is used. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the α-amylase and its variants (mutants) or expressing the desired α-amylase.

"Liquefaction" or "liquefy" means a process by which starch is converted to shorter chain and less viscous dextrins. Generally, this process involves gelatinization of starch simultaneously with or followed by the addition of α-amylase.

According to the present invention, an α-amylase is provided that is the expression product of a mutated DNA sequence encoding an α-amylase, the mutated DNA sequence being derived from a precursor α-amylase by the deletion or substitution of one or more asparagine residues. Also provided is a nucleic acid molecule (DNA) which encodes an amino acid sequence comprising at least a part of the α-amylase provided by the present invention, expression systems incorporating such DNA including vectors and phages, host cells transformed with such DNA, and anti-sense strands of DNA corresponding to the DNA molecule which encodes the amino acid sequence. Similarly, the present invention includes a method for producing an α-amylase by expressing the DNA incorporated on an expression system which has been transformed into a host cell. The α-amylase of the invention may be used in liquefaction of starch, as an ingredient in detergents, in food processing, in textile processing, or in any other application in which α-amylase activity is useful.

The α-amylases according to the present invention comprise an amino acid sequence which is derived from the amino acid sequence of a precursor α-amylase. The precursor α-amylases include naturally occurring α-amylases and recombinant α-amylases. The amino acid sequence of the α-amylase mutant is derived from the precursor α-amylase amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is generally of the precursor DNA sequence which encodes the amino acid sequence of the precursor α-amylase rather than manipulation of the precursor α-amylase enzyme per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein and in commonly owned U.S. Pat. Nos. 4,760,025 and 5,185,258, incorporated herein by reference.

The α-amylases according to the present invention are derived from a precursor amylase. The precursor α-amylase is produced by any source capable of producing α-amylase. Suitable sources of α-amylases are prokaryotic or eukaryotic organisms, including fungi, bacteria, plants or animals. Preferably, the precursor α-amylase is produced by a Bacillus; more preferably, by Bacillus licheniformis, Bacillus amyloliquefaciens or Bacillus stearothermophilus; most preferably, the precursor α-amylase is derived from Bacillus licheniformis.

Homologies have been found between almost all endo-amylases sequenced to date, ranging from plants, mammals, and bacteria (Nakajima et al., Appl. Microbiol. Biotechnol., vol. 23, pp. 355-360 (1986); Rogers, Biochem. Biophys. Res. Commun., vol. 128, pp. 470-476 (1985); Janecek, Eur. J. Biochem., vol. 224, pp. 519-524 (1994)). There are four areas of particularly high homology in certain Bacillus amylases, as shown in FIG. 5, wherein the underlined sections designate the areas of high homology. Sequence alignments have also been used to map the relationship between Bacillus endo-amylases (Feng et al., J. Molec. Evol., vol. 35, pp. 351-360 (1987)). The relative sequence homology between Bacillus stearothermophilus and Bacillus licheniformis amylase is about 66% and that between Bacillus licheniformis and Bacillus amyloliquefaciens amylases is about 81%, as determined by Holm et al., Protein Engineering, vol. 3, No. 3, pp. 181-191 (1990). While sequence homology is important, it is generally recognized that structural homology is also important in comparing amylases or other enzymes. For example, structural homology between fungal amylases and bacterial amylase has been suggested and, therefore, fungal amylases are encompassed within the present invention.

Among others, residues corresponding to asparagine residues in α-amylase are identified herein for deletion or substitution. Thus, specific residues such as N188 refer to an amino acid position number (i.e., +188) which references the number assigned to the mature Bacillus licheniformis α-amylase sequence illustrated in FIG. 4. The invention, however, is not limited to the mutation of the particular mature α-amylase of Bacillus licheniformis but extends to precursor α-amylases containing amino acid residues at positions which are equivalent to the particular identified residue in Bacillus licheniformis α-amylase. A residue of a precursor α-amylase is equivalent to a residue of Bacillus licheniformis α-amylase if it is either homologous (i.e., corresponds in position for either the primary or tertiary structure) or analogous to a specific residue or portion of that residue in Bacillus licheniformis α-amylase (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally).

In order to establish homology to primary structure, the amino acid sequence of a precursor α-amylase is directly compared to the Bacillus licheniformis α-amylase primary sequence and particularly to a set of residues known to be invariant to all α-amylases for which sequences are known (see e.g., FIG. 7). It is possible also to determine equivalent residues by tertiary structure analysis of the crystal structures reported for porcine pancreatic α-amylase (Buisson et al., EMBO Journal, vol. 6, pp. 3909-3916 (1987); Qian et al., Biochemistry, vol. 33, pp. 6284-6294 (1994); Larson et al., J. Mol. Biol., vol. 235, pp. 1560-1584 (1994)); Taka-amylase A from Aspergillus oryzae (Matsuura et al., J. Biochem. (Tokyo), vol. 95, pp. 697-702 (1984)); and an acid α-amylase from A. niger (Boel et al. Biochemistry, vol. 29, pp. 6244-6249 (1990)), with the former two structures being similar, and for barley α-amylase (Vallee et al., J. Mol. Biol., vol. 236, pp. 368-371(1994); Kadziola, J. Mol. Biol., vol. 239, pp.104-121 (1994)). Although there have been some preliminary studies published (Suzuki et al, J. Biochem., vol. 108, pp. 379-381 (1990); Lee et al., Arch. Biochem. Biophys, vol. 291, pp. 255-257 (1991); Chang et al, J. Mol. Biol., vol. 229, pp. 235-238 (1993); Mizuno et al., J. Mol. Biol., vol. 234, pp. 1282-1283 (1993)), there is only a published structure for Bacillus licheniformis α-amylase (Machius et al., J. Mol. Biol. vol. 246, pp. 545-549 (1995)). However, several researchers have predicted common super-secondary structures between glucanases (MacGregor et al., Biochem. J., vol. 259, pp. 145-152 (1989)) and within α-amylases and other starch-metabolising enzymes (Jaspersen, J. Prot. Chem. vol. 12, pp. 791-805 (1993); MacGregor, Starke, vol. 45, pp. 232-237 (1993)); and sequence similarities between enzymes with similar super-secondary structures to α-amylases (Janecek, FEBS Letters, vol. 316, pp. 23-26 (1993); Janecek et al., J. Prot. Chem., vol. 12, pp. 509-514 (1993)). A structure for the Bacillus stearothermophilus enzyme has been modeled on that of Taka-amylase A (Holm et al., Protein Engineering, vol. 3, pp. 181-191 (1990)). The four highly conserved regions shown in FIG. 7 contain many residues thought to be part of the active-site (Matsuura et al., J. Biochem. (Tokyo), vol. 95, pp. 697-702 (1984); Buisson et al., EMBO Journal, vol. 6, pp. 3909-3916 (1987); Vihinen et al., J. Biochem., vol. 107, pp. 267-272 (1990)) including His+105; Arg+229; Asp+231; His+235; Glu+261 and Asp+328 under the Bacillus licheniformis numbering system.

Preferably, the deleted or substituted asparagine residue is at a position corresponding to N188 in Bacillus licheniformis. Where it is desired to alter the thermostability of the α-amylase, the asparagine substitution may be any other amino acid, including any of the 20 naturally occurring amino acids. Preferably, the deletion or substitution corresponds to N188S or N188T in Bacillus licheniformis. Also preferably, the α-amylase further comprises the deletion or substitution of a methionine or tryptophan residue.

The α-amylases according to the present invention exhibit altered performance characteristics providing desirable and unexpected results which are useful in the various applications for which α-amylases are commonly used. For example, α-amylases according to the present invention which exhibit altered performance characteristics at low pH, including improved thermostability, improved pH stability and/or improved oxidative stability, are useful in low pH liquefaction of starch. Enhanced thermostability will be useful in extending the shelf life of products which incorporate them. Enhanced oxidative stability or improved performance is particularly desirable in cleaning products, and for extending the shelf life of α-amylase in the presence of bleach, perborate, percarbonate or peracids used in such cleaning products. To the contrary, reduced thermal stability or oxidative stability may be useful in industrial processes which require the rapid and efficient quenching of amylolytic activity.

The α-amylase of the present invention is especially useful in starch processing and particularly in starch liquefaction. Conditions present during commercially desirable liquefaction processes characteristically include low pH, high temperature and potential oxidation conditions requiring α-amylases exhibiting improved low pH performance, improved thermal stability and improved oxidative stability. Accordingly, α-amylases according to the present invention which are particularly useful in liquefaction exhibit improved performance at a pH of less than about 6, preferably less than about 5.5, and most preferably between about 5.0 and 5.5. Additionally, α-amylases according to the present invention which exhibit increased thermal stability at temperatures of between about 80-120° C., and preferably between about 100-110° C., and increased stability in the presence of oxidants will be particularly useful. Preferably, the α-amylase according to the present invention which is used in liquefaction, in addition to deletion or substitution of an asparagine, further comprises a deletion or substitution at one or more residues corresponding to M15, V128, H133, W138, S187, M197 and/or A209 in Bacillus licheniformis. In a more preferred embodiment, α-amylase used in starch liquefaction according to the present invention comprises a deletion or substitution corresponding to position N188. Most preferably, the amylase comprises a substitution corresponding to M15T/N188S, M15L/N188S, M15T/H133Y/N188S, M15T/H133Y/N188S/A209V, M15T/N188S/A209V, M15T/V128E/H133Y/N188S, M15T/S187D/N188S, M15T/H13 or M15T/H 133Y/A209V in Bacillus licheniformis.

Additional components known by those skilled in the art to be useful in liquefaction, including, for example, antioxidants, calcium, ions, salts or other enzymes such as endoglycosidases, cellulases, proteases, lipases or other amylase enzymes may be added depending on the intended reaction conditions. For example, combinations of the α-amylase according to the present invention with α-amylases from other sources may provide unique action profiles which find particular use under specific liquefaction conditions. In particular, it is contemplated that the combination of the α-amylase according to the present invention with α-amylase derived from Bacillus stearothermophilus will provide enhanced liquefaction at pH values below 5.5 due to complementary action patterns. A preferred embodiment where the process involves the liquefaction of dry milled starch for ethanol production comprises α-amylase derived from Bacillus stearothermophilus and α-amylase according to the present invention having a substitution corresponding to M15T/N188S or M15L/N188S in Bacillus licheniformis.

During liquefaction, starch, specifically granular starch slurries from either a wet or dry milled process, is treated with an α-amylase of the present invention according to known liquefaction techniques. Generally, in the first step of the starch degradation process, the starch slurry is gelatinized by heating at a relatively high temperature (between about 80° C. and about 110° C.). After the starch slurry is gelatinized, it is liquefied using an α-amylase.

In another embodiment of the present invention there are provided detergent compositions in either liquid, gel or granular form, which comprise the α-amylase according to the present invention. Such detergent compositions will particularly benefit from the addition of an α-amylase according to the present invention which has increased thermal stability to improve shelf-life or increased oxidative stability such that the α-amylase has improved resistance to bleach or peracid compounds commonly present in detergents. Thus, α-amylase according to the present invention may be advantageously formulated into known powdered, liquid or gel detergents having a pH of between about 6.5 and about 12.0. A preferred embodiment of the present invention further comprises the deletion or substitution of a methionine residue or a tryptophan residue, for example M15, M197 or W138 as described in commonly assigned U.S. patent application Ser. Nos. 08/289,351 and 08/409,771, the disclosures of which are incorporated by reference; substitution at M133Y as described in PCT Publication No. WO 91/00353; or substitution at A209 as described in DeClerck, et al., J. Biol. Chem., vol. 265, pp. 15481-15488 (1990). Also preferably, an α-amylase according to the present invention used in detergent compositions comprises a deletion or substitution at position N188. Detergent compositions comprising the α-amylase according to the present invention may further include other enzymes such as endoglycosidases, cellulases, proteases, lipases or other amylase enzymes, particularly α-amylase derived from Bacillus stearothermophilus, as well as additional ingredients as generally known in the art.

Embodiments of the present invention which comprise a combination of the α-amylase according to the present invention with protease enzymes preferably include oxidatively stable proteases such as those described in U.S. Re. Pat. No. 34,606, incorporated herein by reference, as well as commercially available enzymes such as DURAZYM (Novo Nordisk), MAXAPEM (Gist-brocades) and PURAFECT® OxP (Genencor International, Inc.). Methods for making such protease mutants (oxidatively stable proteases), and particularly such mutants having a substitution for the methionine at a position equivalent to M222 in Bacillus amyloliquefaciens, are described in U.S. Re. Pat. No. 34,606.

An additional embodiment of the present invention comprises DNA encoding an α-amylase according to the present invention and expression vectors comprising such DNA. The DNA sequences may be expressed by operably linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate host according to well known techniques. A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, include segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as the various known plasmids and phages useful for this purpose. In addition, any of a wide variety of expression control sequences are generally used in these vectors. For example, Applicants have discovered that a preferred expression control sequence for Bacillus transformants is the aprE signal peptide derived from Bacillus subtilis.

A wide variety of host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, various fungi, yeast and animal cells. Preferably, the host expresses the α-amylase of the present invention extracellularly to facilitate purification and downstream processing. Expression and purification of the mutant α-amylase of the invention may be effected through art-recognized means for carrying out such processes.

The improved α-amylases according to the present invention provide several important advantages when compared to wild type Bacillus α-amylases. For example, one advantage is the increased activity found at low pH and high temperatures typical of common starch liquefaction methods. Another advantage is the increased high pH and oxidative stability which facilitates their use in detergents. Another advantage is that a more complete hydrolysis of starch molecules is achieved which reduces residual starch in the processing stream. Yet another advantage is their improved stability in the absence of calcium ion. Yet another advantage is that the addition of equal protein doses of α-amylase according to the invention provide superior performance when compared to wild type Bacillus licheniformis α-amylase due to improvements in both specific activity and stability under stressed conditions. In other words, because of the generally increased stability of the amylases according to the present invention, the increased specific activity on starch of the inventive amylases translates to even greater potential performance benefits of this variant. Under conditions where the wild type enzyme is being inactivated, not only does more of the inventive amylase survive because of its increased stability, but also that which does survive expresses proportionally more activity because of its increased specific activity.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims. Abbreviations used herein, particularly three letter or one letter notations for amino acids are described in Dale, J. W., Molecular Genetics of Bacteria, John Wiley & Sons, (1989) Appendix B.

EXAMPLES Example 1 Construction of Plasmid pHP.BL

The α-amylase gene shown in FIG. 3 was cloned from Bacillus licheniformis NCIB8061 (Gray et al., J. Bacteriology, vol. 166, pp. 635-643 (1986)). The 1.72 kb PstI-SstI fragment, encoding the last three residues of the signal sequence, the entire mature protein and the terminator region, was subcloned into M13mp18. A synthetic terminator was added between the BclI and SstI sites using a synthetic oligonucleotide cassette of the form:

    BcII                                                      SstI     5'-GATCAAAACATAAAAAACCGGCCTTGGCCCCGCCGGTTTTTTATTATTTTTGAGCT-3'                                           (SEQ ID NO:1)     3'     TTTTGTATTTTTTGGCCGGAACCGGGGCGGCCAAAAAATAATAAAAAC     5'                                           (SEQ ID NO:2)

designed to contain the Bacillus amyloliquefaciens subtilisin transcriptional terminator (Wells et al., Nucleic Acid Research, vol. 11, pp. 7911-7925 (1983)).

The pBLapr plasmid was constructed carrying the gene for the Bacillus licheniformis α-amylase. As illustrated in FIG. 7, pBLapr comprises a 6.1 kb plasmid including the ampicillin resistance gene from pBR322 and the chloramphenicol resistance gene from pC194, the aprE promoter and the gene encoding for the Bacillus licheniformis α-amylase ("BL AA"). The aprE promoter was constructed from a 660 bp HindIII-PstI fragment encoding for the promoter and signal sequence of the Bacillus subtilis alkaline protease. The PstI site was removed, and an SfiI site added close to the aprE/BL AA junction. The BL AA gene comprises the 1720 bp PstI-Sstl fragment described above. In the work described herein, pBLapr was constructed with an SfiI site adjacent to the 5' end of the start of the coding sequence for the mature amylase gene. Specifically, the 5' end of the pBLapr construction was subcloned on an EcoRI-SstII fragment from pBLapr into M13BM20 (Boehringer Mannheim) to obtain a coding-strand template for the mutagenic oligonucleotide below:

    5'-CCC ATT AAG ATT GGC CGC CTG GGC CGA CAT GTT GCT GG-3'                                       (SEQ ID NO:3)

This primer introduced an SfiI site (indicated by underlining) which allowed correct forms to be screened for by the presence of this unique restriction site. Subcloning the EcoRI-SstII fragment back into the pBLapr vector gave a version of the plasmid containing an SfiI site.

Plasmid pHP13 (Haima et al., Mol. Gen. Genet., vol. 209, pp. 335-342 (1987)) (FIG. 6) was digested with restriction enzymes EcoRI and HindIII and the resulting vector purified on a polyacrymide gel and then eluted. Plasmid pBLapr was digested with HindIII, Asp718 and in a separate incubation with Asp718, EcoRI and gel purified. Two bands, HindIII -Asp718 (1203 bp) and Asp718-EcoRI (1253 bp) were gel purified, eluted from the gel and ligated into the vector by a 3-way ligation, to give plasmid pHP.BL, the plasmid used in expression of the α-amylase (FIG. 8).

Example 2 Construction of Plasmid Encoding α-Amylase Comprising Substitutions for Asparagine 188

A series of mutagenic primers encoding for substitutions of Asn188 ("N188") with each of the naturally occurring amino acids were synthesized and are shown in FIG. 1 (SEQ ID NOS:4-22). The α-amylase gene mutations encoding for these changes were made by PCR, according to the procedure summarized in FIG. 9, using the PCR primers shown in FIG. 2 (SEQ ID NOS:23-32).

Step (1)

The mutagenic primers were used as templates for the PCR primers PCR A+ and PCR B- resulting in a lengthened (61 bp) double stranded DNA. Each contained a different amino acid replacement at position 188, and all except N188M contained a different restriction site. Initially the PCR primers were annealed at 35° C. for five minutes followed by a one minute DNA extension with taq polymerase at 75° C. The double stranded DNA was then melted at 95° C. for one minute, followed by the annealing and extension steps. Melting, annealing and extension continued for a total of 30 cycles.

Step (2)

DNA upstream and downstream of position 188 were made in separate PCR reactions. The template was pBLapr, and the PCR primers were LAAfs5 (SEQ ID NO:27) and PCR A- (SEQ ID NO:24) for upstream; and PCR B+ (SEQ ID NO:25) and PCR Cla-SalI (SEQ ID NO:28) for downstream DNA. The DNA was melted at 95° C. for one minute, annealed at 45° C. for three minutes and elongated at 68° C. for 3 minutes. The upstream portion is 290 bp and downstream is 498 bp. This procedure was repeated for 18 cycles using pfu polymerase. The same PCR procedure was used in steps (3) and (4).

Step (3)

The upstream portion of DNA described in step (2) was attached to the double stranded mutagenic primers described in step (1). Primers LAAfs5 (SEQ ID NO:27) and PCR B- (SEQ ID NO:26) were used. As the result of primer design there is a 24 bp overlap between these templates allowing for the attachment of the two pieces of DNA.

Step (4)

The downstream portions of DNA described in Step (2) and the product of Step (3) were attached to give the final product. A 24 bp overlap between the two PCR products allows for the attachment. Primers used were LAAfs5 (SEQ ID NO:27) and PCR ClaI-SalI (SEQ ID NO:28).

Step (5)

Unique restriction sites, Asp718 and BssHII, are located upstream and downstream, respectively, of the 188 site. The final PCR product is digested with Asp718 and BssHII, the 333 bp fragment isolated by polyacrylamide gel electrophoresis and subcloned into the pHP.BL vector to obtain pHP.N188X.

Mutations were confirmed by dideoxy sequencing (Sanger et al., Proc. Natl. Acad. Sci. U.S.A., vol. 74, pp. 5463-5467 (1977)).

With reference to the DNA sequence and numbering system used in FIG. 3, the codon encoding for the +188 amino acid position is at base pairs 812-814. PCR primers A+ and A- correspond to base pairs 784-807. PCR primers B+ and B- correspond to base pairs 821-844. The 5' end of PCR primer LAAfs5 corresponds to base pair 518. The 5' end of PCR primer PCR ClaI-SalI corresponds to base pair 1317. The Asp718 site corresponds to base pair 724. The BssHII site corresponds to base pair 1053.

Example 3 Construction of Plasmid Encoding Mutations at M15 and N188

A pBLapr plasmid having threonine substituted for methionine at amino acid 15 was constructed according to U.S. patent application Ser. No. 08/194,664 (PCT Publication No. WO 94/18314). This plasmid (pBLaprM15T) was digested with SfiI and Asp718, and the 477 base pair fragment subcloned into pHP.BL to create pHP.M15T. In a manner analogous to that described above, Example 1, pHP.M15T was digested with Asp718 and BssHII, gel purified and eluted from the gel. The 333 base pair fragment comprising Asp718 to BssHII and the fragment from pHP.N188S were then subcloned into pHP.M15T to give plasmid pHP.M15T/N188S. In an analogous manner, starting with plasmids pBL aprM15L and pHP.N188Y, the plasmid pHP.M15L/N188Y was constructed.

Example 4 Transformation of Plasmids into Bacillus subtilis, Expression and Purification of Mutant α-Amylase

α-Amylase was expressed in Bacillus subtilis after transformation with the plasmids described in Examples 1-3. pHP13 is a plasmid able to replicate in E. coli and in Bacillus subtilis. Plasmids containing different variants were constructed using E. coli strain MM294, the plasmids isolated and then transformed into Bacillus subtilis as described in Anagnostopoulos et al., J. Bacter., vol. 81, pp. 741-746 (1961). The Bacillus strain had been deleted for two proteases (Δapr, Δnpr) (see e.g., Ferrari et al., U.S. Pat. No. 5,264,366) and for amylase (ΔamyE) (see e.g., Stahl et al., J. Bacter., vol. 158, pp. 411-418 (1984)). The bacillus strain expressing M15L/N188Y was found to form larger zones of clearing than the strain expressing M15L on agar plates containing 1% insoluble starch indicating increased amylolytic activity. After transformation, the sacU(Hy) mutation (Henner et al., J. Bacter., vol., 170, pp. 296-300 (1988)) was introduced by PBS-1 mediated transduction (Hoch, J. Bact., vol. 154, pp. 1513-1515 (1983)).

Secreted amylases were routinely recovered from Bacillus subtilis cultures as follows: The culture supernatant was adjusted to 20% saturated ammonium sulfate and stirred for one hr. at 4° C. After centrifugation, the resultant supernatant was adjusted to 70% saturated ammonium sulfate and stirred for one hr. at 4° C. After centrifugation of the supernatant, the resultant pellet was re-dissolved in 50 mM sodium acetate, pH 6.0, 5 mM calcium chloride, and sterile filtered.

Example 5 Assay for Determining α-Amylase Activity

Soluble Substrate Assay

A rate assay was developed based on an end-point assay kit supplied by Megazyme (Aust.) Pty. Ltd. A vial of substrate (p-nitrophenyl maltoheptaoside, BPNPG7) was dissolved in 10 ml of sterile water followed by a 1:4 dilution in assay buffer (50 mM maleate buffer, pH 6.7, 5 mM calcium chloride, 0.002% Tween20). Assays were performed by adding 10 μl of amylase to 790 μl of the substrate in a cuvette at 25° C. Rates of hydrolysis were measured as the rate of change of absorbance at 410 nm, after a delay of 75 seconds. The assay was linear up to rates of 0.2 absorption units/min.

α-Amylase protein concentration was measured using the standard Bio-Rad Assay (Bio-Rad Laboratories) based on the method of Bradford, Anal. Biochem., vol. 72, p. 248 (1976) using bovine serum albumin standards.

Starch Hydrolysis Assay

α-Amylase activity on starch was determined through an assay which depends on the ability of starch to form a blue colored complex with iodine and the disappearance of this color when starch is hydrolyzed to shorter dextrin molecules. The α-amylase activity was defined in terms of the digestion time required to produce a color change denoting a definite state of dextrination of the starch.

Reagents used were as follows:

Phosphate buffer--Potassium dihydrogen phosphate (340 g) and sodium hydroxide (25.3 g) were dissolved in water and diluted to ˜two liters. The buffer was cooled to room temperature and the pH was adjusted to 6.2±0.1. The buffer was diluted to two liters in a volumetric flask.

Starch substrate--Ten grams (dry substance) of soluble lintner starch were suspended in 50 ml of water and washed into ˜300 ml of boiling water. The suspension was again brought to boiling and was boiled for five minutes with constant stirring. The starch solution was cooled with constant stirring to room temperature and 125 ml of phosphate buffer was added. The solution was diluted to 500 ml with water. The starch substrate was made fresh daily.

Stock iodine solution--Iodine crystals (5.5 g) and potassium iodide (11.0 g) were dissolved in water and were volumetrically diluted to 250 ml. The solution was kept from light.

Dilute iodine solution--Potassium iodide (20 g) and two ml of stock iodine solution were dissolved in water and diluted volumetrically to 500 ml. The solution was made fresh daily.

Enzyme diluting solution--Calcium chloride (11.1 g) was dissolved in four liters of water. Water used for all reagents was either distilled or deionized.

An α-amylase sample was diluted to between 10-15 LU/ml (as defined below) with enzyme diluting solution. For many commercial α-amylase preparations a suitable dilution was found to be 2000 fold. Five milliliter aliquots of dilute iodine solution were dispensed into 13×100 mm test tubes and 10 ml of starch substrate was placed in a 23×200 mm test tube. All tubes were placed in the 30° C. water bath. A Hellige comparator equipped with a special α-amylase color disc (catalog number 620-s5) was used to make readings. Five milliliters of diluted enzyme (also at 30° C.) were mixed with the starch substrate and timing was begun. At appropriate time intervals, for example one minute intervals early in the reaction and 15 second intervals later in the reaction, one ml aliquots of the enzyme-substrate mixture were transferred to a tube containing the dilute iodine solution. The starch iodine solution was mixed and transferred to a 13 mm precision square tube and the color was compared with the standard α-amylase color disc in the Hellige comparator. When the time of the end point was approached, samples were taken at 0.25 minute intervals.

The time required for the colors of the samples and the color disc to match were recorded and the activity (in liquefons per gram or ml) was calculated according to the formula: ##EQU1## Where: LU=liquefon unit

V=volume of enzyme (5 ml or grams)

t=dextrinization time (minutes)

D=dilution factor: dilution volume divided by ml or g of enzyme diluted.

Mutant α-amylases according to the invention prepared as in Examples 1-4 were tested for their specific activity on starch and soluble substrate. The results, as shown in Table 1, illustrate that mutant amylase according to the invention provides a superior activity profile in comparison with the AA20 wild type α-amylase on both substrates.

                  TABLE 1     ______________________________________     Specific Activity Of Certain α-Amylases On Soluble     Substrate And Starch As Percentage Of Wild Type Activity                   Soluble Substrate                               Starch Hydrolysis     α-AMYLASE                   Assay       Assay     ______________________________________     Spezyme ® AA20                   100         100     M15T/N188S    212         166     ______________________________________

Example 6 Starch Liquefaction Conditions--Determination of Liquefied Starch DE (Dextrose Equivalent)

Starch liquefaction was performed using a reactor composed of 50 feet of 0.24 inch diameter (0.21 inch i.d.) stainless steel tubing bent into an approximately 10 inch diameter coil ˜5.5 inches high. The coil was equipped with an 11.5 inch in-line static mixer (Cole-Parmer #G-04669-60) mounted ˜4 feet from the anterior end. The posterior end of the coil was equipped with a Swagelok in-line adjustable pressure relief value (#SS-4CA-3) set at a cracking pressure of about 20 psi. Starch slurry was fed to the coil at a rate of ˜70 ml/minute with a piston metering pump. The temperature of the reactor coil was held at 105.5° C. by immersion of the reactor in a glycerol-water bath. Temperature in the bath was maintained using a circulating heater/temperature controller (Fisher Scientific model 7305).

Starch liquefaction at the pilot scale was typically performed using a Hydroheater M 103-M steam jet equipped with a 2.5 liter delay coil behind the mixing chamber and a terminal back pressure valve. Starch was fed to the jet by a Moyno pump and steam was supplied by a 150 psi steam line, reduced to 90-100 psi. Temperature probes were installed just after the Hydroheater jet and just before the back pressure valve. Starch was introduced into the jet at about 350 ml/min. The jet temperature was held at 105-107° C. Samples of starch were transferred from the jet cooker to a 95° C. second stage liquefaction and held for 90 minutes.

Granular starch was obtained from a corn wet miller and used within two days. The starch was diluted to a desired solids level of about 30-35% dry solids with deionized water and the pH was adjusted with 2.5% NaOH or 6% HCl as required. Calcium was added in the form of CaCl₂.2H₂ O. Typical liquefaction conditions were:

    ______________________________________     Starch       30%-35% solids     Calcium      40-60 ppm (30 ppm added)     pH           5.0-6.0     α-amylase                  12-14 LU/g of carbohydrate (dry basis)     ______________________________________

Samples of starch were transferred from the reactor to a 95° C. second stage liquefaction bath and held for 90 minutes. The degree of starch liquefaction was measured immediately after the second stage liquefaction by determining the dextrose equivalent (DE) of the sample according to the method described in the Standard Analytical Methods of the Member Companies of the Corn Refiners Association, Inc., sixth ed., Analytical Procedure Committee (1980).

Example 7 Comparison of M15T/N188S and Wild Type α-Amylase in Liquefaction at 105.5° C.

α-Amylase comprising the substitution M15T/N188S made as per Examples 1-4 was compared with wild type α-amylase derived from Bacillus licheniformis (Spezyme® AA20, available commercially from Genencor International, Inc.) in liquefaction at 105.5° C. As shown in Table 2, the mutant enzymes provided significantly increased performance in jet-liquefaction of starch, especially at low pH. Pilot scale liquefaction was performed with a primary stage liquefaction at 105.5° C. and a secondary stage liquefaction at 95° C. Amylase was added at 12 LU/g of carbohydrate (dry basis).

                  TABLE 2     ______________________________________     Comparative Liquefaction Performance Of α-Amylases At 105.5°     C.     AMYLASE             pH    DE     ______________________________________     Spezyme ® AA20  6.0    9.85     (Average of Two     Runs)     G11                 6.0   12.2     (Average of Four     Runs)     Spezyme ® AA20  5.5   5.4     G11                 5.5   8.7     (Average of Two     Runs)     Spezyme ® AA20  5.2   1.8     G11                 5.2   3.0     ______________________________________

Example 8 Comparison of M15T/N188S and Wild Type α-Amylase in Liquefaction at 107.0° C.

α-Amylase comprising substitution M15T/N188S made as per Examples 1-4 was compared with wild type α-amylase derived from Bacillus licheniformis (Spezyme® AA20, available commercially from Genencor International, Inc.) in liquefaction at 107° C. As shown in Table 3, the mutant enzymes provided significantly increased performance in jet-liquefaction of starch especially at low pH, as shown by the DE value, during liquefaction processes. Pilot scale liquefaction was performed with a primary stage liquefaction at 107° C. and a secondary stage liquefaction at 95° C. Amylase was added at 12 LU/g or carbohydrate (dry basis).

                  TABLE 3     ______________________________________     Comparative Liquefaction Performance of α-Amylase At 107°     C.     VARIANT           pH    DE     ______________________________________     AA20              6.0   7.4     G11               6.0   11.6     AA20              5.5   3.5     G11               5.5   6.0     AA20              5.2   0     G11               5.2   1.1     ______________________________________

Example 9 Statistical Analysis of Liquefaction Results for Mutant and Wild Type α-Amylase

The relative liquefaction performance of Spezyme® AA20 and the M15T/N188S variant were extensively explored in a statistical design experiment. Using the "X-STAT" program, Version 2.0 (Copyright, Wiley Scientific and Technical Software, John Wiley & Sons, New York, (1992)), a Box-Behnken factorial experiment was designed; varying the primary liquefaction temperature from 106° C. to 110° C., the liquefaction pH from pH 5.3 to pH 6.0, and the total calcium level in the starch substrate from 30 ppm to 90 ppm. The data in Tables 4 and 5 which formed the basis of this experiment was generated in 15 pilot scale liquefactions each, using 12 LU/gram dry solid substrate of Spezyme® AA20 and M15T/N188S. The data was then fitted to quadratic models. For the M15T/N188S variant, the data fitted the equation DE=842.41+28.374×pH-17.557×Temperature+1.5005×Calcium concentration+1.6243 (pH×Temperature)-0.081506 (pH×Calcium concentration)-0.0092099 (Temperature×Calcium concentration)-16.841 (pH)² +0.038379 (Temperature)² -0.000124 (Calcium concentration)² with a standard error about the regression of 1.313 and an explained variation about the mean (R)² of 93.99%. For Spezyme® AA20, the data was fitted to the equation DE=-652.0+(132.35×pH)+(4.716×Temperature)+(1.3989×Calcium concentration)-0.050515 (pH×Temperature)-0.019603 (pH×Calcium concentration)-0.011118 (Temperature×Calcium concentration)-10.206 (pH)² +0.02104 (Temperature)² -0.000522 (Calcium concentration)². With a standard error about the regression of 0.5772 and an explained variation about the mean (R²) of 98.69%, these equations were used to prepare curves plotting calculated DE vs. pH, vs. Calcium concentration vs. Temperature. Two dimensional representations of that data at 107° C. and 60 ppm Ca+ are illustrated in FIGS. 10-12 respectively. As shown in FIGS. 10-12, the mutant amylase outperforms the wild type amylase by enabling more efficient liquefaction of starch at lower pH, lower levels of calcium and higher temperature.

                  TABLE 4     ______________________________________                                   Observed                                   Dextrose             Temperature   Calcium Equivalent     pH      Celsius       ppm     M15T/N188S     ______________________________________     6.00    110.2         60.0    9.8     6.00    105.9         60.0    11.7     5.30    110.2         60.0    2.1     5.30    106.5         60.0    8.1     6.00    108.0         90.0    11.3     6.00    107.6         30.0    10.3     5.30    108.4         90.0    5.9     5.30    108.5         30.0    1.7     5.65    110.2         90.0    9.5     5.65    109.8         30.0    9.9     5.65    106.0         90.0    11.9     5.65    105.5         30.0    9.9     5.65    107.8         60.0    9.5     5.65    108.1         60.0    9.6     6.00    108.3         60.0    11.6     ______________________________________

                  TABLE 5     ______________________________________                                  Observed                                  Dextrose            Temperature   Calcium Equivalent     pH     Celsius       ppm     Spezyme ® AA20     ______________________________________     6.00   110.0         60      7.4     6.00   106.2         60      9.9     5.30   109.7         60      0.6     5.30   105.8         60      2.9     6.00   108.3         90      8.5     6.00   108.4         30      7.8     5.30   108.6         90      1.2     5.30   107.5         30      0.4     5.65   110.0         90      4.1     5.65   109.5         30      4.0     5.65   106.8         90      8.6     5.65   106.0         30      6.4     5.65   107.8         60      6.1     5.65   109.0         60      5.9     5.65   109.0         60      5.9     ______________________________________

While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions and changes may be made without departing from the spirit and scope thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims, including equivalents thereof.

Example 10 Preparation and Testing of Additional Mutant Alpha-Amylases for Thermal Stability

Mutant alpha-amylases were prepared having substitutions at one or more of positions V128E, H133Y, S187D and/or A209V generally according to the procedures provided in Examples 1-4 except that appropriate PCR primers were provided to effect the desired mutations. Amylases were purified to a point where wild type Bacillus licheniformis α-amylase showed a specific activity of 1087 LU/mg protein. Protein concentration was determined by absorption at 278 nm, using a Molar Extinction coefficient of wild type enzyme of 143,255 M⁻¹ cm⁻¹.

Thermal inactivation rates for the various mutants were measured according to the following procedure. Amylase stock solutions were dialysed extensively into 20 mM ammonium acetate, 4 mM CaCl₂ pH 6.5. For measurement of stability, this stock was diluted >50 fold into 50 mM ammonium acetate, 5mM CaCl₂, 0.02% Tween 20 pH 5.0 to a final concentration of between 30 and 50 μg/ml. Six 100 μl aliquots were put into eppendorf tubes and placed into a water bath at 83° C. The eppendorf tubes were removed at regular, measured intervals of between 30 seconds and 5 minutes and placed on ice to stop the inactivation. The residual activity was assayed using a soluble substrate as described in Example 5. The natural log of the activity was plotted against time of incubation, and the rate constant for inactivation obtained from the slope of the straight line. Results for various mutants are provided in Table 6.

                  TABLE 6     ______________________________________                    Inactivation                    Rate      Half Life                                       Improvement                    Constant, (In2/k)  Over Wild     Amylase        k(min.sup.-1)                              (min)    Type     ______________________________________     Wild Type      1.2       0.56     1.0     M15T/N188S     0.81      0.86     1.5     M15L/N188S     0.76      0.91     1.6     M15T/H133Y     0.39      1.8      3.2     M15T/H133Y/N188S                    0.31      2.2      4.0     M15T/N188S/A209V                    0.27      2.5      4.5     M15T/H133Y/N188S/A209V                    0.054     13       23     ______________________________________

Example 11 Low pH Liquefaction Performance of Variant α-Amylases

α-Amylase comprising substitutions M15T/N188S or M15T/H133Y/N188S were made as per Examples 1-4 and 10 and compared in liquefaction studies as per Example 6. Liquefaction was performed at 105.5° C. with a secondary hold of 90 minutes at 95° C. under conditions including 94 ppm SO₂ with amylase at a concentration of 16 LU/g of carbohydrate (dry basis). The results are provided in Table 7 below.

                  TABLE 7     ______________________________________     Amylase            pH     DE     ______________________________________     M15T/N188S         5.50   11.6     M15T/H133Y/N188S   5.50   13.9     M15T/N188S         5.35   7.8     M15T/H133Y/N188S   5.35   10.0     M15T/N188S         5.20    3.2     M15T/H133Y/N188S   5.20    5.0     ______________________________________

Example 12 Low pH Liquefaction Performance of M15T/V128E/H133Y/N188S, M15T/H133Y/N188S and M15T/N188S at Varying Calcium Levels

α-Amylase comprising various substitutions were made as per Examples 14 and 10 and compared in liquefaction studies as per Example 6. Liquefaction was performed at 105.5° C. under conditions including a pH of 5.50, 95 ppm SO₂ with amylase at a concentration of 12 LU/g of carbohydrate (dry basis). The results are provided in Table 8 below.

                  TABLE 8     ______________________________________     Amylase            Calcium Added                                   DE     ______________________________________     M15/V128E/H133Y/N188S                        44         11.8     M15T/H133Y/N188S   44         12.4     M15T/N188S         44         9.9     M15T/V128E/H133Y/N188S                         0         8.9     M15T/H133Y/N188S    0         7.6     M15T/N188S          0         4.9     ______________________________________

Example 13 Low pH Liquefaction Performance of M15T/H133Y and M15T/H133Y/A209V at Varying pH Levels

α-Amylase comprising various substitutions were made as per Examples 1-4 and 10 and compared in liquefaction studies as per Example 6. Liquefaction was performed at 105.5° C. under conditions including 98 ppm SO₂ with amylase at a concentration of 19 LU/g of carbohydrate (dry basis). Dried corn starch (Clinton Brand 106-B Pearl cornstarch, ADM Corn Processing, Clinton, Iowa) was slurried with deionized water (˜23 kg in ˜50 liters) and allowed to hydrate for 16 hours. The results are provided in Table 9 below.

                  TABLE 9     ______________________________________     Amylase              pH     DE     ______________________________________     M15T/H133Y/N188S     5.00    6.8     M15T/H133Y/N188S/A209V                          5.00   10.0     M15T/H133Y/N188S     5.25   11.6     M15T/H133Y/N188S/A209V                          5.25   13.2     M15T/H133Y/N188S     5.50   14.3     M15T/H133Y/N188S/A209V                          5.50   15.9     ______________________________________

Example 14 Improved Liquefaction Performance of Variant α-Amylase Compared to Wild Type

α-Amylase comprising substitution at M15T/S187D/N188S were made as per Examples 1-4 and 10 and compared to wild type in liquefaction studies as per Example 6. Dried corn starch (Clinton Brand 106-B Pearl cornstarch, ADM Corn Processing, Clinton, Iowa) was slurried with deionized water (˜23 kg in ˜50 liters) and allowed to hydrate for 16 hours. Liquefaction was performed at 105.6° C. with equal protein levels of amylase at 9.0 μg amylase/g carbohydrate (dry basis) (3.1 mg amylase/liter of 35% dry solid starch slurry). Due to the specific activity benefit derived from the mutant alpha amylase, the activity of the amylases was 11 LU/g carbohydrate (dry basis) for the wild type amylase and 24 LU/g carbohydrate for the mutant. Measured activities showed that the mutant amylase had an activity increase of 410% of the wild type on heptamaltose and 219% of the wild type on starch. The liquefaction results are provided in Table 10 below.

                  TABLE 10     ______________________________________     Amylase            pH     DE     ______________________________________     Wild Type          6.00    8.9     M15T/S187D/N188S   6.00   11.2     ______________________________________

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 40     - (2) INFORMATION FOR SEQ ID NO: 1:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 56 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - GATCAAAACA TAAAAAACCG GCCTTGGCCC CGCCGGTTTT TTATTATTTT TG - #AGCT       56     - (2) INFORMATION FOR SEQ ID NO: 2:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 48 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                48CCGG CGGGGCCAAG GCCGGTTTTT TATGTTTT     - (2) INFORMATION FOR SEQ ID NO: 3:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 38 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #     38           GCCT GGGCCGACAT GTTGCTGG     - (2) INFORMATION FOR SEQ ID NO: 4:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          ACTG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 5:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          CCAG AAAATGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 6:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          AGAG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 7:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          CTCG AGAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 8:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          GCCG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 9:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          GGAG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 10:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          AAGG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 11:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          CAGG AAAATGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 12:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          CAGG AAAATGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 13:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          CATG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 14:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          GAAG AGAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 15:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          GAGG AGAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 16:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          TATG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 17:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          TGCG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 18:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          TTCG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 19:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          ATCG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 20:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          ATGG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 21:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          TGGG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 22:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 37 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #22:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #      37          TCTG AAAACGGCAA CTATGAT     - (2) INFORMATION FOR SEQ ID NO: 23:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #23:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                24TGGG AAGT     - (2) INFORMATION FOR SEQ ID NO: 24:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #24:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                24CCTT TCCT     - (2) INFORMATION FOR SEQ ID NO: 25:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #25:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                24TGAT GTAT     - (2) INFORMATION FOR SEQ ID NO: 26:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #26:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                24TAGT TGCC     - (2) INFORMATION FOR SEQ ID NO: 27:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #27:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #21                TTAA C     - (2) INFORMATION FOR SEQ ID NO: 28:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 22 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #28:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                 22AAA AG     - (2) INFORMATION FOR SEQ ID NO: 29:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #29:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                24AAAC TGGG     - (2) INFORMATION FOR SEQ ID NO: 30:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #30:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                24ACAT GATT     - (2) INFORMATION FOR SEQ ID NO: 31:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #31:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                24ATTG GCAG     - (2) INFORMATION FOR SEQ ID NO: 32:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #32:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     #                24ACCG TAAA     - (2) INFORMATION FOR SEQ ID NO: 33:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 1968 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: double               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA (genomic)     #33:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - AGCTTGAAGA AGTGAAGAAG CAGAGAGGCT ATTGAATAAA TGAGTAGAAA GC - #GCCATATC       60     - GGCGCTTTTC TTTTGGAAGA AAATATAGGG AAAATGGTAC TTGTTAAAAA TT - #CGGAATAT      120     - TTATACAACA TCATATGTTT CACATTGAAA GGGGAGGAGA ATCATGAAAC AA - #CAAAAACG      180     - GCTTTACGCC CGATTGCTGA CGCTGTTATT TGCGCTCATC TTCTTGCTGC CT - #CATTCTGC      240     - AGCAGCGGCG GCAAATCTTA ATGGGACGCT GATGCAGTAT TTTGAATGGT AC - #ATGCCCAA      300     - TGACGGCCAA CATTGGAAGC GTTTGCAAAA CGACTCGGCA TATTTGGCTG AA - #CACGGTAT      360     - TACTGCCGTC TGGATTCCCC CGGCATATAA GGGAACGAGC CAAGCGGATG TG - #GGCTACGG      420     - TGCTTACGAC CTTTATGATT TAGGGGAGTT TCATCAAAAA GGGACGGTTC GG - #ACAAAGTA      480     - CGGCACAAAA GGAGAGCTGC AATCTGCGAT CAAAAGTCTT CATTCCCGCG AC - #ATTAACGT      540     - TTACGGGGAT GTGGTCATCA ACCACAAAGG CGGCGCTGAT GCGACCGAAG AT - #GTAACCGC      600     - GGTTGAAGTC GATCCCGCTG ACCGCAACCG CGTAATTTCA GGAGAACACC TA - #ATTAAAGC      660     - CTGGACACAT TTTCATTTTC CGGGGCGCGG CAGCACATAC AGCGATTTTA AA - #TGGCATTG      720     - GTACCATTTT GACGGAACCG ATTGGGACGA GTCCCGAAAG CTGAACCGCA TC - #TATAAGTT      780     - TCAAGGAAAG GCTTGGGATT GGGAAGTTTC CAATGAAAAC GGCAACTATG AT - #TATTTGAT      840     - GTATGCCGAC ATCGATTATG ACCATCCTGA TGTCGCAGCA GAAATTAAGA GA - #TGGGGCAC      900     - TTGGTATGCC AATGAACTGC AATTGGACGG TTTCCGTCTT GATGCTGTCA AA - #CACATTAA      960     - ATTTTCTTTT TTGCGGGATT GGGTTAATCA TGTCAGGGAA AAAACGGGGA AG - #GAAATGTT     1020     - TACGGTAGCT GAATATTGGC AGAATGACTT GGGCGCGCTG GAAAACTATT TG - #AACAAAAC     1080     - AAATTTTAAT CATTCAGTGT TTGACGTGCC GCTTCATTAT CAGTTCCATG CT - #GCATCGAC     1140     - ACAGGGAGGC GGCTATGATA TGAGGAAATT GCTGAACGGT ACGGTCGTTT CC - #AAGCATCC     1200     - GTTGAAATCG GTTACATTTG TCGATAACCA TGATACACAG CCGGGGCAAT CG - #CTTGAGTC     1260     - GACTGTCCAA ACATGGTTTA AGCCGCTTGC TTACGCTTTT ATTCTCACAA GG - #GAATCTGG     1320     - ATACCCTCAG GTTTTCTACG GGGATATGTA CGGGACGAAA GGAGACTCCC AG - #CGCGAAAT     1380     - TCCTGCCTTG AAACACAAAA TTGAACCGAT CTTAAAAGCG AGAAAACAGT AT - #GCGTACGG     1440     - AGCACAGCAT GATTATTTCG ACCACCATGA CATTGTCGGC TGGACAAGGG AA - #GGCGACAG     1500     - CTCGGTTGCA AATTCAGGTT TGGCGGCATT AATAACAGAC GGACCCGGTG GG - #GCAAAGCG     1560     - AATGTATGTC GGCCGGCAAA ACGCCGGTGA GACATGGCAT GACATTACCG GA - #AACCGTTC     1620     - GGAGCCGGTT GTCATCAATT CGGAAGGCTG GGGAGAGTTT CACGTAAACG GC - #GGGTCGGT     1680     - TTCAATTTAT GTTCAAAGAT AGAAGAGCAG AGAGGACGGA TTTCCTGAAG GA - #AATCCGTT     1740     - TTTTTATTTT GCCCGTCTTA TAAATTTCTT TGATTACATT TTATAATTAA TT - #TTAACAAA     1800     - GTGTCATCAG CCCTCAGGAA GGACTTGCTG ACAGTTTGAA TCGCATAGGT AA - #GGCGGGGA     1860     - TGAAATGGCA ACGTTATCTG ATGTAGCAAA GAAAGCAAAT GTGTCGAAAA TG - #ACGGTATC     1920     #              1968CCTG AGACTGTGAC GGATGAATTG AAAAAGCT     - (2) INFORMATION FOR SEQ ID NO: 34:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 483 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     #34:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Ph - #e Glu Trp Tyr Met Pro     #                15     - Asn Asp Gly Gln His Trp Lys Arg Leu Gln As - #n Asp Ser Ala Tyr Leu     #            30     - Ala Glu His Gly Ile Thr Ala Val Trp Ile Pr - #o Pro Ala Tyr Lys Gly     #        45     - Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Ty - #r Asp Leu Tyr Asp Leu     #    60     - Gly Glu Phe His Gln Lys Gly Thr Val Arg Th - #r Lys Tyr Gly Thr Lys     #80     - Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu Hi - #s Ser Arg Asp Ile Asn     #                95     - Val Tyr Gly Asp Val Val Ile Asn His Lys Gl - #y Gly Ala Asp Ala Thr     #           110     - Glu Asp Val Thr Ala Val Glu Val Asp Pro Al - #a Asp Arg Asn Arg Val     #       125     - Ile Ser Gly Glu His Leu Ile Lys Ala Trp Th - #r His Phe His Phe Pro     #   140     - Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Tr - #p His Trp Tyr His Phe     145                 1 - #50                 1 - #55                 1 -     #60     - Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Le - #u Asn Arg Ile Tyr Lys     #               175     - Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Se - #r Asn Glu Asn Gly Asn     #           190     - Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Ty - #r Asp His Pro Asp Val     #       205     - Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Ty - #r Ala Asn Glu Leu Gln     #   220     - Leu Asp Gly Phe Arg Leu Asp Ala Val Lys Hi - #s Ile Lys Phe Ser Phe     225                 2 - #30                 2 - #35                 2 -     #40     - Leu Arg Asp Trp Val Asn His Val Arg Glu Ly - #s Thr Gly Lys Glu Met     #               255     - Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Le - #u Gly Ala Leu Glu Asn     #           270     - Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Va - #l Phe Asp Val Pro Leu     #       285     - His Tyr Gln Phe His Ala Ala Ser Thr Gln Gl - #y Gly Gly Tyr Asp Met     #   300     - Arg Lys Leu Leu Asn Gly Thr Val Val Ser Ly - #s His Pro Leu Lys Ser     305                 3 - #10                 3 - #15                 3 -     #20     - Val Thr Phe Val Asp Asn His Asp Thr Gln Pr - #o Gly Gln Ser Leu Glu     #               335     - Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Al - #a Tyr Ala Phe Ile Leu     #           350     - Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Ty - #r Gly Asp Met Tyr Gly     #       365     - Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Al - #a Leu Lys His Lys Ile     #   380     - Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Al - #a Tyr Gly Ala Gln His     385                 3 - #90                 3 - #95                 4 -     #00     - Asp Tyr Phe Asp His His Asp Ile Val Gly Tr - #p Thr Arg Glu Gly Asp     #               415     - Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Le - #u Ile Thr Asp Gly Pro     #           430     - Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gl - #n Asn Ala Gly Glu Thr     #       445     - Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pr - #o Val Val Ile Asn Ser     #   460     - Glu Gly Trp Gly Glu Phe His Val Asn Gly Gl - #y Ser Val Ser Ile Tyr     465                 4 - #70                 4 - #75                 4 -     #80     - Val Gln Arg     - (2) INFORMATION FOR SEQ ID NO: 35:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 511 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     #35:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Le - #u Leu Thr Leu Leu Phe     #                15     - Ala Leu Ile Phe Leu Leu Pro His Ser Ala Al - #a Ala Ala Ala Asn Leu     #            30     - Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Ty - #r Met Pro Asn Asp Gly     #        45     - His Trp Lys Arg Leu Gln Asn Asp Ser Ala Ty - #r Leu Ala Glu His Gly     #    60     - Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Ly - #s Gly Thr Ser Gln Ala     #80     - Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr As - #p Leu Gly Glu Phe His     #                95     - Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Th - #r Lys Gly Glu Leu Gln     #           110     - Ser Ala Ile Lys Ser Leu His Ser Arg Asp Il - #e Asn Val Tyr Gly Asp     #       125     - Val Val Ile Asn His Lys Gly Gly Ala Asp Al - #a Thr Glu Asp Val Thr     #   140     - Ala Val Glu Val Asp Pro Ala Asp Arg Asn Ar - #g Val Ile Ser Gly Glu     145                 1 - #50                 1 - #55                 1 -     #60     - His Leu Ile Lys Ala Trp Thr His Phe His Ph - #e Pro Gly Arg Gly Ser     #               175     - Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr Hi - #s Phe Asp Gly Thr Asp     #           190     - Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Ty - #r Lys Phe Gln Gly Lys     #       205     - Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gl - #y Asn Tyr Asp Tyr Leu     #   220     - Met Tyr Ala Asp Ile Asp Tyr Asp His Pro As - #p Val Ala Ala Glu Ile     225                 2 - #30                 2 - #35                 2 -     #40     - Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Le - #u Gln Leu Asp Gly Phe     #               255     - Arg Leu Asp Ala Val Lys His Ile Lys Phe Se - #r Phe Leu Arg Asp Trp     #           270     - Val Asn His Val Arg Glu Lys Thr Gly Lys Gl - #u Met Phe Thr Val Ala     #       285     - Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Gl - #u Asn Tyr Leu Asn Lys     #   300     - Thr Asn Phe Asn His Ser Val Phe Asp Val Pr - #o Leu His Tyr Gln Phe     305                 3 - #10                 3 - #15                 3 -     #20     - His Ala Ala Ser Thr Gln Gly Gly Gly Tyr As - #p Met Arg Lys Leu Leu     #               335     - Asn Gly Thr Val Val Ser Lys His Pro Leu Ly - #s Ser Val Thr Phe Val     #           350     - Asp Asn His Asp Thr Gln Pro Gly Gln Ser Le - #u Glu Ser Thr Val Gln     #       365     - Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Il - #e Leu Thr Arg Glu Ser     #   380     - Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Ty - #r Gly Thr Lys Gly Asp     385                 3 - #90                 3 - #95                 4 -     #00     - Ser Gln Arg Glu Ile Pro Ala Leu Lys His Ly - #s Ile Glu Pro Ile Leu     #               415     - Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gl - #n His Asp Tyr Phe Asp     #           430     - His His Asp Ile Val Gly Trp Thr Arg Glu Gl - #y Asp Ser Ser Val Ala     #       445     - Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gl - #y Pro Gly Gly Ala Lys     #   460     - Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Gl - #u Thr Trp His Asp Ile     465                 4 - #70                 4 - #75                 4 -     #80     - Thr Gly Asn Arg Ser Glu Pro Val Val Ile As - #n Ser Glu Gly Trp Gly     #               495     - Glu Phe His Val Asn Gly Gly Ser Val Ser Il - #e Tyr Val Gln Arg     #           510     - (2) INFORMATION FOR SEQ ID NO: 36:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 520 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     #36:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - Met Arg Gly Arg Gly Asn Met Ile Gln Lys Ar - #g Lys Arg Thr Val Ser     #                15     - Phe Arg Leu Val Leu Met Cys Thr Leu Leu Ph - #e Val Ser Leu Pro Ile     #            30     - Thr Lys Thr Ser Ala Val Asn Gly Thr Leu Me - #t Gln Tyr Phe Glu Trp     #        45     - Tyr Thr Pro Asn Asp Gly Gln His Trp Lys Ar - #g Leu Gln Asn Asp Ala     #    60     - Glu His Leu Ser Asp Ile Gly Ile Thr Ala Va - #l Trp Ile Pro Pro Ala     #80     - Tyr Lys Gly Leu Ser Gln Ser Asp Asn Gly Ty - #r Gly Pro Tyr Asp Leu     #                95     - Tyr Asp Leu Gly Glu Phe Gln Gln Lys Gly Th - #r Val Arg Thr Lys Tyr     #           110     - Gly Thr Lys Ser Glu Leu Gln Asp Ala Ile Gl - #y Ser Leu His Ser Arg     #       125     - Asn Val Gln Val Tyr Gly Asp Val Val Leu As - #n His Lys Ala Gly Ala     #   140     - Asp Ala Thr Glu Asp Val Thr Ala Val Glu Va - #l Asn Pro Ala Asn Arg     145                 1 - #50                 1 - #55                 1 -     #60     - Asn Gln Glu Thr Ser Glu Glu Tyr Gln Ile Ly - #s Ala Trp Thr Asp Phe     #               175     - Arg Phe Pro Gly Arg Gly Asn Thr Tyr Ser As - #p Phe Lys Trp His Trp     #           190     - Tyr His Phe Asp Gly Ala Asp Trp Asp Glu Se - #r Arg Lys Ile Ser Arg     #       205     - Ile Phe Lys Phe Arg Gly Glu Gly Lys Ala Tr - #p Asp Trp Glu Val Ser     #   220     - Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Ty - #r Ala Asp Val Asp Tyr     225                 2 - #30                 2 - #35                 2 -     #40     - Asp His Pro Asp Val Val Ala Glu Thr Lys Ly - #s Trp Gly Ile Trp Tyr     #               255     - Ala Asn Glu Leu Ser Leu Asp Gly Phe Arg Il - #e Asp Ala Ala Lys His     #           270     - Ile Lys Phe Ser Phe Leu Arg Asp Trp Val Gl - #n Ala Val Arg Gln Ala     #       285     - Thr Gly Lys Glu Met Phe Thr Val Ala Glu Ty - #r Trp Gln Asn Asn Ala     #   300     - Gly Lys Leu Glu Asn Tyr Leu Asn Lys Thr Se - #r Phe Asn Gln Ser Val     305                 3 - #10                 3 - #15                 3 -     #20     - Phe Asp Val Pro Leu His Phe Asn Leu Gln Al - #a Ala Ser Ser Gln Gly     #               335     - Gly Gly Tyr Asp Met Arg Arg Leu Leu Asp Gl - #y Thr Val Val Ser Arg     #           350     - His Pro Glu Lys Ala Val Thr Phe Val Glu As - #n His Asp Thr Gln Pro     #       365     - Gly Gln Ser Leu Glu Ser Thr Val Gln Thr Tr - #p Phe Lys Pro Leu Ala     #   380     - Tyr Ala Phe Ile Leu Thr Arg Glu Ser Gly Ty - #r Pro Gln Val Phe Tyr     385                 3 - #90                 3 - #95                 4 -     #00     - Gly Asp Met Tyr Gly Thr Lys Gly Thr Ser Pr - #o Lys Glu Ile Pro Ser     #               415     - Leu Lys Asp Asn Ile Glu Pro Ile Leu Lys Al - #a Arg Lys Glu Tyr Ala     #           430     - Tyr Gly Pro Gln His Asp Tyr Ile Asp His Pr - #o Asp Val Ile Gly Trp     #       445     - Thr Arg Glu Gly Asp Ser Ser Ala Ala Lys Se - #r Gly Leu Ala Ala Leu     #   460     - Ile Thr Asp Gly Pro Gly Gly Ser Lys Arg Me - #t Tyr Ala Gly Leu Lys     465                 4 - #70                 4 - #75                 4 -     #80     - Asn Ala Gly Glu Thr Trp Tyr Asp Ile Thr Gl - #y Asn Arg Ser Asp Thr     #               495     - Val Lys Ile Gly Ser Asp Gly Trp Gly Glu Ph - #e His Val Asn Asp Gly     #           510     - Ser Val Ser Ile Tyr Val Gln Lys     #       520     - (2) INFORMATION FOR SEQ ID NO: 37:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 548 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     #37:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - Val Leu Thr Phe His Arg Ile Ile Arg Lys Gl - #y Trp Met Phe Leu Leu     #                15     - Ala Phe Leu Leu Thr Ala Ser Leu Phe Cys Pr - #o Thr Gly Arg His Ala     #            30     - Lys Ala Ala Ala Pro Phe Asn Gly Thr Met Me - #t Gln Tyr Phe Glu Trp     #        45     - Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Ly - #s Val Ala Asn Glu Ala     #    60     - Asn Asn Leu Ser Ser Leu Gly Ile Thr Ala Le - #u Ser Leu Pro Pro Ala     #80     - Tyr Lys Gly Thr Ser Arg Ser Asp Val Gly Ty - #r Gly Val Tyr Asp Leu     #                95     - Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Th - #r Val Arg Thr Lys Tyr     #           110     - Gly Thr Lys Ala Gln Tyr Leu Gln Ala Ile Gl - #n Ala Ala His Ala Ala     #       125     - Gly Met Gln Val Tyr Ala Asp Val Val Phe As - #p His Lys Gly Gly Ala     #   140     - Asp Gly Thr Glu Trp Val Asp Ala Val Glu Va - #l Asn Pro Ser Asp Arg     145                 1 - #50                 1 - #55                 1 -     #60     - Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gl - #n Ala Trp Thr Lys Phe     #               175     - Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Se - #r Phe Lys Trp Arg Trp     #           190     - Tyr His Phe Asp Gly Val Asp Trp Asp Glu Se - #r Arg Lys Leu Ser Arg     #       205     - Ile Tyr Lys Phe Arg Gly Ile Gly Lys Ala Tr - #p Asp Trp Glu Val Asp     #   220     - Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Ty - #r Ala Asp Leu Asp Met     225                 2 - #30                 2 - #35                 2 -     #40     - Asp His Pro Glu Val Val Thr Glu Leu Lys As - #n Trp Gly Lys Trp Tyr     #               255     - Val Asn Thr Thr Asn Ile Asp Gly Phe Arg Le - #u Asp Gly Leu Lys His     #           270     - Ile Lys Phe Ser Phe Phe Pro Asp Trp Leu Se - #r Tyr Val Arg Ser Gln     #       285     - Thr Gly Lys Pro Leu Phe Thr Val Gly Glu Ty - #r Trp Ser Tyr Asp Ile     #   300     - Asn Lys Leu His Asn Tyr Ile Thr Lys Thr As - #n Gly Thr Met Ser Leu     305                 3 - #10                 3 - #15                 3 -     #20     - Phe Asp Ala Pro Leu His Asn Lys Phe Tyr Th - #r Ala Ser Lys Ser Gly     #               335     - Gly Ala Phe Asp Met Arg Thr Leu Met Thr As - #n Thr Leu Met Lys Asp     #           350     - Gln Pro Thr Leu Ala Val Thr Phe Val Asp As - #n His Asp Thr Asn Pro     #       365     - Ala Lys Arg Cys Ser His Gly Arg Pro Trp Ph - #e Lys Pro Leu Ala Tyr     #   380     - Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pr - #o Cys Val Phe Tyr Gly     385                 3 - #90                 3 - #95                 4 -     #00     - Asp Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pr - #o Ser Leu Lys Ser Lys     #               415     - Ile Asp Pro Leu Leu Ile Ala Arg Arg Asp Ty - #r Ala Tyr Gly Thr Gln     #           430     - His Asp Tyr Leu Asp His Ser Asp Ile Ile Gl - #y Trp Thr Arg Glu Gly     #       445     - Val Thr Glu Lys Pro Gly Ser Gly Leu Ala Al - #a Leu Ile Thr Asp Gly     #   460     - Ala Gly Arg Ser Lys Trp Met Tyr Val Gly Ly - #s Gln His Ala Gly Lys     465                 4 - #70                 4 - #75                 4 -     #80     - Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser As - #p Thr Val Thr Ile Asn     #               495     - Ser Asp Gly Trp Gly Glu Phe Lys Val Asn Gl - #y Gly Ser Val Ser Val     #           510     - Trp Val Pro Arg Lys Thr Thr Val Ser Thr Il - #e Ala Arg Pro Ile Thr     #       525     - Thr Arg Pro Trp Thr Gly Glu Phe Val Arg Tr - #p His Glu Pro Arg Leu     #   540     - Val Ala Trp Pro     545     - (2) INFORMATION FOR SEQ ID NO: 38:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 32 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     #38:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - Met Lys Gln Gln Lys Arg Leu Thr Ala Arg Le - #u Leu Thr Leu Leu Phe     #                15     - Ala Leu Ile Phe Leu Leu Pro His Ser Ala Al - #a Ala Ala Ala Asn Leu     #            30     - (2) INFORMATION FOR SEQ ID NO: 39:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 33 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     #39:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - Met Arg Ser Lys Thr Leu Trp Ile Ser Leu Le - #u Phe Ala Leu Thr Leu     #                15     - Ile Phe Thr Met Ala Phe Ser Asn Met Ser Al - #a Gln Ala Ala Gly Lys     #            30     - Ser     - (2) INFORMATION FOR SEQ ID NO: 40:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 32 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     #40:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:     - Met Arg Ser Lys Thr Leu Trp Ile Ser Leu Le - #u Phe Ala Leu Thr Leu     #                15     - Ile Phe Thr Met Ala Phe Ser Asn Met Ser Al - #a Gln Ala Ala Asn Leu     #            30     __________________________________________________________________________ 

We claim:
 1. An α-amylase that is the expression product of a mutated DNA sequence encoding an α-amylase, the mutated DNA sequence being derived from a precursor α-amylase by substitution corresponding to N188T.
 2. The α-amylase according to claim 1, wherein said substitution or substitution further comprises the deletion or substitution of a methionine or tryptophan residue.
 3. The α-amylase according to claim 2, wherein said substitution or substitution of said methionine or tryptophan residue comprises a substitution or deletion corresponding to M15, W138 or M197 in Bacillus licheniformis.
 4. The α-amylase according to claim 1 wherein said substitution or substitution further comprises the deletion or substitution of a residue corresponding to V129, H133, S187 or A209 in Bacillus licheniformis.
 5. An α-amylase that is the expression product of a mutated DNA sequence encoding an α-amylase, the mutated DNA sequence being derived from a precursor α-amylase by a substitution corresponding to M15T/N188S, M15L/N188S, M15T/H133Y/N188S, M15T/H133Y/N188S/A209V, M15T/N188S/A209V, M15T/V128E/H133Y/N188S, M15T/S187D/N188S, M15T/N188T, M15L/N188T, M15T/H133Y/N188T, M15T/H133Y/N188T/A209V, M15T/N188T/A209V. M15T/V128E/H133Y/N188T, M15T/S187D/N188T in Bacillus licheniformis.
 6. The α-amylase according to claim 3, wherein said substitution of said methionine residues comprises a substitution corresponding to M15T, W138Y or M197T in Bacillus licheniformis.
 7. The α-amylase according to claim 1, wherein said precursor α-amylase is derived from Bacillus.
 8. The α-amylase according to claim 7, wherein said precursor α-amylase is derived from Bacillus licheniformis.
 9. A DNA encoding the α-amylase according to claim
 1. 10. A DNA encoding the α-amylase according to claim
 3. 11. A DNA encoding the α-amylase according to claim
 4. 12. A DNA encoding the α-amylase according to claim
 5. 13. An expression vector comprising the DNA of claim
 9. 14. An expression vector comprising the DNA of claim
 10. 15. An expression vector comprising the DNA of claim
 11. 16. An expression vector comprising the DNA of claim
 12. 17. A host cell transformed with the expression vector of claim
 13. 18. A host cell transformed with the expression vector of claim
 14. 19. A host cell transformed with the expression vector of claim
 15. 20. A host cell transformed with the expression vector of claim
 16. 21. An α-amylase according to claims 1, 4 or 5 having enhanced low pH performance.
 22. A detergent composition comprising the α-amylase according to claims 1, 4 or
 5. 23. The detergent composition according to claim 22, wherein said detergent is useful in laundering soiled fabric.
 24. The detergent composition according to claim 22, wherein said detergent is useful in washing soiled dishes.
 25. A method of liquefying starch comprising the steps of:(a) preparing a solution comprising starch polymer; (b) combining with said solution of starch polymer the α-amylase according to claim 1 under conditions and for a time suitable to liquefy said starch polymer.
 26. The method of claim 25, wherein said α-amylase comprises the α-amylase according to claim
 2. 27. The method of claim 25, wherein said α-amylase comprises the α-amylase according to claim
 3. 28. The method of claim 25, wherein said α-amylase comprises the α-amylase according to claim
 4. 29. The method of claim 25, wherein said α-amylase comprises the α-amylase according to claim
 5. 30. The method of claim 25, wherein said α-amylase comprises the α-amylase according to claim
 6. 31. The method of claim 25, wherein said α-amylase comprises the α-amylase according to claim
 7. 32. The method of claim 25, wherein said α-amylase comprises the α-amylase according to claim
 8. 